U.S. patent number 6,294,331 [Application Number 09/130,078] was granted by the patent office on 2001-09-25 for methods for assessing genetic and phenotypic markers by simultaneous multicolor visualization of chromogenic dyes using brightfield microscopy and spectral imaging.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services. Invention is credited to Anton H. N. Hopman, Merryn V. E. MacVille, Thomas Ried.
United States Patent |
6,294,331 |
Ried , et al. |
September 25, 2001 |
Methods for assessing genetic and phenotypic markers by
simultaneous multicolor visualization of chromogenic dyes using
brightfield microscopy and spectral imaging
Abstract
The present invention is directed to an improved method for
detecting a genetic marker in a biological sample comprising
contacting the biological sample with a nucleic acid probe linked
to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the genetic marker in the
biological sample. The present invention also provides an improved
method for detecting a phenotypic marker in a biological sample
comprising contacting the biological sample with a compound
comprising a detectable moiety, whereby the compound associates
with the phenotypic marker and whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
Inventors: |
Ried; Thomas (Bethesda, MD),
MacVille; Merryn V. E. (The Hague, NL), Hopman; Anton
H. N. (Eijsden, NL) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
26734234 |
Appl.
No.: |
09/130,078 |
Filed: |
August 7, 1998 |
Current U.S.
Class: |
435/6.1; 359/368;
435/7.1; 435/7.92; 536/24.3 |
Current CPC
Class: |
C12Q
1/6841 (20130101); C12Q 1/6841 (20130101); C12Q
2565/601 (20130101); C12Q 2563/179 (20130101); C12Q
2563/107 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C12N 015/11 ();
G02B 021/12 () |
Field of
Search: |
;435/6,7.1,7.92
;536/24.3 ;359/368 |
Foreign Patent Documents
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|
|
|
|
|
|
WO 97/21979 |
|
Jun 1997 |
|
WO |
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WO 97/22848 |
|
Jun 1997 |
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WO |
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Other References
Malik et al. Fourier transform multipixel spectroscopy for
quantitative cytology. J. Microscopy vol. 182 pp. 133-140, 1995.*
.
Schrock, et al., "Multicolor Spectral Karyotyping of Human
Chromosomes", Science, vol. 273, pp. 494-497, Jul., 1996. .
Speel, et al., "Cytochemical detection systems for in situ
hybridization, and the combination with immunocytochemistry. `Who
is still afraid of Red, Green and blue?`", Histochemical Journal,
27:833-858, 1995. .
Garini, et al., "Spectral karyotyping", Bioimaging, 4:65-72,
1996..
|
Primary Examiner: Brusca; John S.
Attorney, Agent or Firm: Needle & Rosenberg
Parent Case Text
This application claims the benefit of U.S. Provisional Application
No. 60/055,439, filed Aug. 8, 1997.
Claims
What is claimed is:
1. A method for detecting a genetic marker in a biological sample
comprising:
a) contacting the biological sample with a nucleic acid probe
linked to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety;
b) obtaining a spectral image of the biological sample using
brightfield microscopy; and
c) detecting the presence of the chromogenic dye, thereby detecting
the genetic marker in the biological sample.
2. The method of claim 1, wherein the biological sample is
contacted with nucleic acid probes having at least two different
specificities.
3. The method of claim 1, wherein the biological sample is attached
to a substrate.
4. The method of claim 1, wherein step (a) further comprises
contacting the biological sample with a cytological stain.
5. The method of claim 1, wherein step (c) further comprises
distinguishing the cytological stain from the detectable
moiety.
6. The method of claim 1, wherein the genetic marker is selected
from the group consisting of a centromere, a telomere, a general
genetic loci, a specific genetic loci, a chromosome band, a
chromosome-specific loci, a chromosome fragment, and a whole
chromosome.
7. The method of claim 1, wherein the detectable moiety comprises a
hapten.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods of detecting genetic and
phenotypic markers in biological samples using spectral imaging and
brightfield microscopy to detect the presence of chromogenic
dyes.
2. Background Art
In cytopathological diagnostic laboratories, cytological specimens
are routinely stained with permanent dyes such as hematoxylin and
eosin and for decades, pathologists have based their diagnosis of
disease on cyto- and histological features as seen under a light
microscope. Unlike fluorescent dyes, permanent dyes do not fade or
bleach so that second opinion diagnosis, re-examination of archived
material and even retrospective studies, can be performed. Thus,
for routine cytopathological diagnostic purposes, fluorescence
microscopy is not preferred for these reasons as well as because of
high auto-fluorescence inherent to the tissue type or which might
be induced by fixation.
Immunohistochemical and in situ hybridization methods have become
increasingly important for research and diagnosis of disease. Also,
multi-parameter cytochemical analysis is required when rare or
unique material is to be studied. Many fluorescent markers with
emission spectra ranging from blue to infra-red have become
available for multi-color detection due to advances made in
conjugation chemistry. Thus, although fluorescence microscopy could
be used for these multi-parameter applications, for the
above-mentioned reasons, it is often not possible to use methods
employing fluorescence.
The present invention overcomes previous shortcomings in the art by
providing methods for analyzing both genetic and phenotypic markers
in a single biological sample through the use of bright field
spectral imaging of chromogenic dyes. Such analyses are valuable in
a variety of clinical applications, such as, for example, the
diagnosis and characterization of cancer and the analysis of
chromosomal aberrations in pre- and post-natal diagnostics.
An important aspect of the present invention that overcomes a
severe limitation in the art is that by using the methods provided
herein, multiple probes, both to genetic and/or phenotypic markers,
and therefore multiple chromogenic dyes can be used in the same
sample and the individual dyes can be distinguished using spectral
imaging, even where the sample has been previously stained with a
cytological stain which otherwise would obscure the signal from the
genetic or phenotypic probes. Using these methods, a pathologist
for example, can stain a tissue sample to observe a general
morphological aspect of cells in the sample, and a geneticist can
subsequently use that stained sample to diagnose cells in the
sample for the presence of a genetic or phenotypic marker, such as
a chromosomal aberration associated with cervical cancer, with much
more clarity, accuracy, ease, and efficiency than using previously
available methods.
SUMMARY OF THE INVENTION
In accordance with the purpose(s) of this invention, as embodied
and broadly described herein, this invention, in one aspect,
relates to the present invention provides an improved method for
detecting a genetic marker in a biological sample comprising
contacting the biological sample with a nucleic acid probe linked
to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the genetic marker in the
biological sample.
The present invention also provides an improved method for
detecting a phenotypic marker in a biological sample comprising
contacting the biological sample with a compound comprising a
detectable moiety, whereby the compound associates with the
phenotypic marker and whereby the detectable moiety can be detected
by the presence of a chromogenic dye associated with the detectable
moiety, obtaining a spectral image of the biological sample using
brightfield microscopy, and detecting the presence of the
chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
Additional advantages of the invention will be set forth in part in
the description which follows, and in part will be obvious from the
description, or may be learned by practice of the invention. The
advantages of the invention will be realized and attained by means
of the elements and combinations particularly pointed out in the
appended claims. It is to be understood that both the foregoing
general description and the following detailed description are
exemplary and explanatory only and are not restrictive of the
invention, as claimed.
The accompanying figures, which are incorporated in and constitute
a part of this specification, illustrate several embodiments of the
invention and together with the description, serve to explain the
principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawings will be provided
by the Patent and Trademark Office upon request and payment of the
necessary fee.
FIG. 1. Triple color spectral imaging of T24 cells, using
chromosome 1, 7 and 15 centromere-specific probes. Targets were
visualized with, respectively, DAB, NF and TMB. Bottom left shows
the raw spectral image of the hybridization signals of one cell.
The colors are a result of an arbitrary RGB look-up table and do
not display the colors as seen through the microscope. Bottom
center shows the spectral image after calculation of absorption
spectra. Constituents that absorb light are displayed in the
complementary color. As a consequence, areas that don't absorb
appear black. Top shows spectra of three pixels located on the
different dyes in an absorption intensity per wavelength diagram.
The absorption peaks are wide apart and the shape of the curves
differ significantly. Bottom right shows the result of a spectrum
based classification whereby pixels with similar spectral
information are assigned the same false color. This classification
is based on the input of the spectra shown in the diagram. The DAB
spots (4) are false-colored with a brown color, TMB (2) with green
and NF (3) with red. The spectrum of the unstained nucleus differed
sufficiently from the background light to be classified in a blue
color.
FIG. 2. T24 cells hybridized for centromere of chromosome 7 and
visualized with NF. The nucleus is stained with hematoxylin. Bottom
left shows the raw spectral image in arbitrary colors. Bottom
center shows the spectral image after calculation of the absorption
spectra displayed in complementary colors. Top shows the spectra of
a few pixels over the spots and the nucleus. The spectra of the
spots is the sum of the spectra for NF and hematoxylin (compare
with NF spectrum in FIG. 1 bottom left), whereas the spectrum of
the nucleus is solely hematoxylin. Based on the average spectra of
the selected pixels, a classification is performed, shown bottom
right. Spots and nuclei are false-colored in red and blue,
respectively.
FIG. 3. Double target in situ hybridization on sperm cells of a
healthy human, using a chromosome X centromere probe visualized
with DAB and a chromosome Y centromere probe visualized with TMB,
combined with histomorphologic staining with DIFF QUIK, a
commercially available eosin derivative stain. The sperm cells
contain a signal either for the X chromosome or the Y chromosome.
Top left shows the raw spectral image in arbitrary display colors.
It was difficult to determine the color by eye through the
microscope, due to weak spots and overall cytoplasmic staining and
overlapping regions as a result of the preparation (smear)
technique. Top center shows the spectral image after calculation of
the absorption spectra displayed in complementary colors. Middle
left shows the spectra of selected pixels for DAB, TMB spots and
DIFF QUIK. The spectra of the spots are mixed with the spectrum of
DIFF QUIK. Bottom left shows the classification based on the
average spectrum of these pixels, false-coloring DAB spots in
brown, TMB spots in green and DIFF QUIK staining above a certain
intensity threshold in blue. A more accurate classification was
achieved when the spectrum of the morphological staining was
eliminated from the spectral image by subtraction or division. Top
right shows the subtracted image. Bottom right shows the
classification based on the average spectrum of these pixels,
displaying just the hybridization spots in false colors brown (DAB)
and green (TMB).
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference
to the following detailed description of preferred embodiments of
the invention and the Example included therein and to the Figures
and their previous and following description.
Before the present compounds, compositions and methods are
disclosed and described, it is to be understood that this invention
is not limited to specific methods, specific nucleic acid probes,
cytological stains, detectable moities, etc., as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting.
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a nucleic acid probe" includes
multiple nucleic acid probe molecules.
The present invention provides methods by which the assessment of
histological or cytological preparations can be combined with
detection of genetic and phenotypic markers by using spectral
imaging.
From a technical point of view, in multi-target labeling format,
color discrimination by eye through a bright field light microscope
is difficult, if not impossible in common situations where: 1) the
staining is weak; 2) signals are small (e.g. they appear as small
punctuate dots); 3) signals lay close together or merge; or 4)
signals overlap in z-direction. In addition, cytological stains can
obscure the color information from the signals.
Color discrimination based on 3-color CCD video images can be
performed by measuring the hue value. Hue values are introduced to
specify colors numerically. Calculation is based on intensities of
red, green and blue light (RGB) as recorded by the separate
channels of the camera. The formulation used for transforming the
RGB values into hue, however, simplifies the data and does not make
reference to the true physical properties of light. In contrast,
spectral imaging analyzes light as the intensity per wavelength,
which is the only quantity by which to describe the color of light
correctly. In addition, spectral imaging provides spatial data,
because it contains spectral information for every pixel in the
image.
Thus, the present invention provides an improved method for
detecting a genetic marker in a biological sample comprising
contacting the biological sample with a nucleic acid probe linked
to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the genetic marker in the
biological sample.
The present invention also provides an improved method for
detecting a phenotypic marker in a biological sample comprising
contacting the biological sample with a compound comprising a
detectable moiety, whereby the compound associates with the
phenotypic marker and whereby the detectable moiety can be detected
by the presence of a chromogenic dye associated with the detectable
moiety, obtaining a spectral image of the biological sample using
brightfield microscopy, and detecting the presence of the
chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
The method of detecting genetic and phenotypic markers in a
biological sample can be used for diagnosing cancer, identifying
types of cancer; determining a prognosis of a cancer, as well as
for detecting, identifying, diagnosing, characterizing and/or
determining a prognosis of a variety of other disease states or
abnormal conditions which can be detected, identified,
characterized, etc., by genetic and/or phenotypic markers in a
biological sample. The method of the present invention can also be
applied to pre- and post-natal diagnostics. For each of these
methods, the biological sample can be prepared as described herein
or by various other methods which are well known in the art for
preparing biological samples for genetic and phenotypic
analyses.
It is further contemplated that the present invention provides
methods for detecting genetic and phenotypic markers in a
biological sample for comparative cytogenetics (i.e., interspecies
studies) and karyotyping. For these methods, the biological sample
can be prepared according the methods described herein or according
to various methods well known in the art for preparing biological
samples for genetic and phenotypic analysis relating to comparative
cytogenetics and karyotyping.
Detection of phenotypic and genetic markers in a biological sample
can be by microscopy, which can be, but is not limited to,
bright-field microscopy, phase contrast microscopy, interference
contrast microscopy, Nomarski contrast microscopy, dark field
microscopy, reflection contrast microscopy, fluorescence
microscopy, infra-red microscopy, or any other type of light
microscopy. Detection can be done by visualizing the biological
sample in the microscope or by recording an image of the biological
sample photographically (e.g., by producing an image on a silver
halide emulsion film which can be developed for visualization or by
recording a digital image of the sample for visualization via an
output device, such as, for example, a computer monitor or as a
computer printout) as well as by any other means by which the
biological sample can be viewed or recorded. The spectral image of
the biological sample can be taken from the sample directly or from
a recorded image of the sample.
The biological sample of this invention can be from any organism
and can be, but is not limited to, embedded tissue sections, frozen
tissue sections, cell preparations, cytological preparations,
exfoliate samples (e.g., sputum), fine needle aspirations, amnion
cells, fresh tissue, dry tissue, and cultured cells or tissue. It
is further contemplated that the biological sample of this
invention can also be whole cells or cell organelles (e.g.,
nuclei). The biological sample can be unfixed or fixed according to
standard protocols widely available in the art and can also be
embedded in a suitable medium for preparation of the sample. For
example, the biological sample can be embedded in paraffin or other
suitable medium (e.g., epoxy or acrylamide) to facilitate
preparation of the biological specimen for the detection methods of
this invention. Furthermore, the biological sample can be embedded
in any commercially available mounting medium, either aqueous or
organic, depending on the chemical properties of the stain or any
specifically developed medium, such as, for example, as designed
for TMB, based on a thin protein layer cross-linked by formaldehyde
to ensure permanent stabilization of the enzyme reaction products
(Speel et al., 1994. A novel triple-color detection procedure for
brightfield microscopy, combining in situ hybridization with
immunocytochemistry." J. Histochem. Cytochem. 42:1299-1307).
The biological sample can be on, supported by, or attached to, a
substrate which facilitates detection of phenotypic or genetic
markers. A substrate of the present invention can be, but is not
limited to, a microscope slide, a culture dish, a culture flask, a
culture plate, a culture chamber, DNA arrays, ELISA plates, as well
as any other substrate now known or developed in the future for
containing or supporting biological samples for analysis according
to the methods of the present invention. The substrate can be of
any material suitable for the purposes of this invention, such as,
for example, glass, plastic, polystyrene, mica and the like. The
substrates of the present invention can be obtained from commercial
sources or prepared according to standard procedures well known in
the art.
The detection of phenotypic and genetic markers in the biological
sample can be combined with the routine assessment of histological
and cytological specimens, generally carried out by staining with
one or more cytological stains and examining the specimens
microscopically. Thus, the present invention provides for
multi-parameter analyses of the same biological sample.
The biological sample of the present invention can be contacted
with one or more cytological stains. The cytological stains used in
the methods of this invention can be, but are not limited to,
hematoxylin, eosin, methyl green, neutral red, DIFF QUIK (Baxter,
The Netherlands), toluidine blue, alcian blue, isamin blue,
methylene blue, sudan black, periodic acid-Schiff reaction (PAS),
Masson's trichrome method, reticulin stain, Van Gieson, Azan,
Giemsa, NissI, silver and gold stains, osmium and chrom alum, as
well as any other cytological stains now known or identified in the
future. The cytological stains of this invention are available from
commercial sources or can be prepared according to standard methods
well known in the art.
The phenotypic markers identified by the methods of this invention
can be, but are not limited to, messenger RNA, gene products,
antigens, antibodies, and proteins, or fragments thereof, which can
be of, for example, tumor suppressor genes, oncogenes and
proliferation markers. Examples of gene products which can be
detected as phenotypic markers can include, but are not limited to,
gene products of p53, retinoblastoma, Ki67, PCNA, nucleolus
organizing regions and cyclins. These phenotypic markers can be
detected by methods well known in the art, including modifications
of the methods described herein to detect nucleic acid probes, such
as binding to the phenotypic marker a detectable molecule such as a
nucleic acid, a hapten, a protein, an antigen, and an antibody, or
fragments thereof.
The compound or compounds comprising a detectable marker which are
used to detect a phenotypic marker, therefore, include any compound
which can bind to, link to, hybridize to, or otherwise associate
with the phenotypic marker. For example, the compound can be an
antibody to a protein or a fragment of a protein, an antibody to a
nucleic acid, an antibody to a ligand or a fragment of a ligand, an
antibody to an antibody or fragment of an antibody (anti-idiotype
antibody), an antibody to any cellular structure or fragment of the
cellular structure, and the like. Alternatively, the compound can
comprise other molecules such as nucleic acids, ligands, haptens,
cell structures, and fragments thereof.
The genetic markers of this invention can be, but are not limited
to, centromeres, telomeres, general or specific loci, chromosome
bands, a chromosome-specific loci, chromosome fragments, and whole
chromosomes, as well as any genetic marker which detects numerical
chromosome alterations or structural chromosome alterations such as
translocations, breakpoints, microdeletions and amplifications. For
detection of these genetic markers, a nucleic acid probe having
complementarity to the nucleotide sequence of the genetic marker is
contacted with the biological sample under conditions whereby
hybridization of the nucleic acid of the genetic marker and the
nucleic acid probe can occur. These conditions can vary, depending
of the biological sample, genetic marker and nucleic acid probe
used for a given application. The hybridization conditions for a
particular application can be determined according to protocols
standard in the art. Examples of various hybridization conditions
are provided in the Examples herein.
The nucleic acid probe of this invention can be a nucleic acid
comprising the nucleotide sequence of a coding strand or its
complementary strand or the nucleotide sequence of a sense strand
or antisense strand. Thus, the probe of this invention can be
either DNA or RNA and can bind either DNA or RNA, or both, in the
biological sample. The probe can be the coding or complementary
strand of a complete gene or gene fragment. The nucleotide sequence
of the probe can be any sequence having sufficient complementarity
to a nucleic acid sequence in the biological sample to allow for
hybridization of the probe to the target nucleic acid in the
biological sample under a desired hybridization condition. Ideally,
the probe will hybridize only to the nucleic acid target of
interest in the sample and will not bind non-specifically to other
non-complementary nucleic acids in the sample or other regions of
the target nucleic acid in the sample. The hybridization conditions
can be varied according to the degree of stringency desired in the
in situ hybridization. For example, if the hybridization conditions
are for high stringency, the probe will bind only to the nucleic
acid sequences in the sample with which it has a very high degree
of complementarity. Low stringency hybridization conditions will
allow for hybridization of the probe to nucleic acid sequences in
the sample which have some complementarity but which are not as
highly complementary to the probe sequence as would be required for
hybridization to occur at high stringency. The hybridization
conditions will vary depending on the biological sample, probe type
and target. An artisan will know how to optimize hybridization
conditions for a particular application of the present method.
Examples of hybridization conditions are described in the Examples
provided herein.
The nucleic acid probe can be commercially obtained or can be
synthesized according to standard nucleotide synthesizing protocols
well known in the art. Alternatively, the probe can be produced by
isolation and purification of a nucleic acid sequence from
biological materials according to methods standard in the art of
molecular biology (Sambrook et al. 1989. Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Pres, Cold
Spring Harbor, N.Y.). The nucleic acid probe can be amplified
according to well known procedure for amplification of nucleic acid
(e.g., polymerase chain reaction). Furthermore, the probe of this
invention can be linked to any of the detectable moieties of this
invention by protocols standard in the art.
It is further contemplated that the present invention also includes
methods for oligonucleotide hybridization wherein the hybridized
oligonucleotide is used as a primer for an enzyme catalyzed
elongation reaction such as in situ PCR and primed in situ labeling
reactions whereby haptenized nucleotides are incorporated in situ.
Additionally included are methods for in situ hybridization,
employing synthetic peptide nucleic acid (PNA) oligonucleotide
probes (Nielsen et al., 1991. "Sequence-selective recognition of
DNA by strand displacement with a thymine-substituted polyamide."
Science 254:1497-1500; Egholm et al., 1993. "PNA hybridizes to
complementary oligonucleotides obeying the Watson-Crick hydrogen
bonding rules." Nature 365:566-568).
The detectable moieties to which the nucleic acid probe of this
invention can be linked to include, but are not limited to, a
hapten, biotin, digoxigenin, fluorescein isothiocyanate (FITC),
dinitrophenyl, amino methyl coumarin acetic acid,
acetylaminofluorene and mercury-sulfhydryl-ligand complexes, as
well as any other molecule or compound which can be linked to a
nucleic acid probe and detected either directly or indirectly
according to the methods described herein.
In one method of detection, the nucleic acid or compound moiety can
be directly detected by linking the detectable moiety to the
nucleic acid probe or compound and another moiety which can
facilitate direct detection, such as an enzyme (e.g., peroxidase,
alkaline phosphatase, glucose oxidase) which produces a colored
reaction product when reacted with a suitable substrate or to
colloidal gold particles or other detectable moieties.
Alternatively, the nucleic acid or compound can be detected
indirectly by the binding of antibodies, antibody fragments or
other ligands, or the reaction of other molecules (e.g., avidin to
detect biotin) with the detectably moiety linked to the nucleic
acid or compound, including for example, enzymes such as
peroxidase, alkaline phosphatase or glucose oxidase for enzymatic
precipitation upon reaction with suitable substrates to produce a
colored reaction product, i.e., a chromogenic dye associated with
the detectable moiety. The enzyme peroxidase can also be used in
conjunction with tyramide-based detection formats.
The antibodies, antibody fragments or ligands can also be linked to
colloidal gold particles for direct detection or subsequently
enhanced with silver for indirect detection. The detectable
moieties of this invention are available from commercial sources or
can be prepared according to standard protocols well known in the
art. Methods for detecting the detectable moieties of the present
invention are common in the art. Protocols for linking probes,
detectable moieties, antibodies, ligands, etc., are also standard
in the art and are readily available to the artisan. Additionally,
the detectable moieties exemplified here can be detected in any
number of alternative detection procedures other than those
listed.
The detectable moiety of this invention can also comprise an
antibody. The antibody can be either monoclonal or polyclonal. The
antibodies of this invention can also include immunoreactive
antibody fragments. The detectable moiety can also comprise a
ligand or any other molecule that can detect the antibody or the
nucleic acid probe.
Antibodies can be made by many well-known methods (See, e.g. Harlow
and Lane, "Antibodies; A Laboratory Manual" Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified
antigen can be injected into an animal, with or without adjuvants,
in an amount and in intervals sufficient to elicit an immune
response. Polyclonal antibodies can be purified directly, or spleen
cells can be obtained from the animal for monoclonal antibody
production. The spleen cells can be fused with an immortal cell
line and the resulting hybridomas can be screened for antibody
secretion. A variety of immunoassay formats can be used to select
antibodies which selectively bind with a particular protein. For
example, solid-phase ELISA immunoassays are routinely used to
select antibodies selectively immunoreactive with a protein. See
Harlow and Lane (1988) for a description of immunoassay formats and
conditions that can be used to characterize antibody binding.
In some instances, it is desirable to prepare monoclonal antibodies
from various hosts, for example, for anti-species antibodies. A
description of techniques for preparing such monoclonal antibodies
may be found in Stites et al., editors, "Basic and Clinical
Immunology," (Lange Medical Publications, Los Altos, Calif., Fourth
Edition) and references cited therein, as well as in Harlow and
Lane (1988).
As described above, the antibody of the present invention can bind
an antigen which is attached to the nucleic acid probe. The
antibody itself can be linked to a detectable moiety, such as an
enzyme, and binding of antibody to an antigen attached to a nucleic
acid probe can thereby be detected directly. Alternatively, the
antibody which binds the antigen which is attached to the nucleic
acid probe can be detected indirectly, by binding a second antibody
which recognizes the first bound antibody as an antigen. The second
antibody can be linked to a detectable moiety, such as an enzyme,
thereby detecting the binding of the first antibody indirectly.
In the present invention, the spectral image of the biological
sample on the substrate can be obtained with a device which
utilizes a common path Sagnac interferometer creating an optical
path difference based on the angle of incident light. An
interferogram is produced showing the light intensities against the
function of the optical path difference. Fourier transformation of
the interferogram recovers the spectrum. An example of this device
is a SD200 Spectracube (Applied Spectral Image, Migdal HaEmek,
Israel). Other methods to measure absorption spectra using light
microscopy can include spectrophotometry, the use of liquid crystal
tunable filters and accusto-optical tunable filters.
The spectral image of the biological sample of this invention can
be analyzed with software designed for spectral image analysis,
such as the SpCube 1.5 program (Applied Spectral Imaging). The
present invention further contemplates software programs dedicated
to the methods of this invention.
The following examples are intended to illustrate, but not limit,
the invention. While the protocols described are typical of those
that might be used, other procedures known to those skilled in the
art may be alternatively employed.
EXAMPLES
Biological Specimens
T24 human bladder cancer cells (ATCC accession number ATCC HTB 4)
were grown on microscope slides under standard cell culture
conditions to 30% confluency. Cells were fixed and pretreated for
in situ hybridization as described (Speel et al., 1994). Human
spermatozoid cells were obtained from a healthy male, fixed and
smeared on a microscopic slide for pretreatment for in situ
hybridization (Martini, E., et al., 1995. Application of different
in situ hybridization detection methods for human sperm analysis."
Hum. Reprod. 10:855-861).
T24 bladder cells were hybridized with centromeric alpha-satellite
probes for chromosome 1, 7, 15 (Oncor, Gaithersburg, Md.) or
combinations thereof . The sperm cells were hybridized with probes
for X- and Y-chromosome specific loci (Oncor, Gaithersburg, Md.).
For single labeling, probes were labeled by nick-translation with
biotin (Boehringer-Mannheim, Germany) and detected with peroxidase
(PO) conjugated to avidin (Vector, USA) or alkaline phosphatase
(AP) conjugated to avidin (Vector), depending on the enzyme
substrate to be used. Diaminobenzidine (DAB), tetra-methylbenzidine
(TMB) and amino-ethyl-carbazole (AEC) were used as substrates for
PO and New Fuchsin (NF), Fast Red (FR) and NBT/BCIP/INT (INT) were
used as substrates for AP. (Substrates were obtained commercially.)
The enzyme reaction produces a precipitate, i.e. a chromogenic dye,
in situ that is visible in a bright field light microscope.
For double labeling, one probe was labeled with biotin and the
other with digoxigenin (Boehringer-Mannheim). Digoxigenin label was
detected with polyclonal anti-digoxigenin antibody conjugated to
either PO or AP (Boehringer-Mannheim). The third label was
introduced by using fluorescein isothiocyanate (FITC) as a hapten
and detected with mouse anti-FITC antibody (Dako, Denmark) and
anti-mouse-PO or anti-mouse-AP antibody (Boehringer). In double and
triple labeling, interspecies cross-reactivity was blocked and
enzyme reactions producing the reporter signals were developed
sequentially (Speel et al., 1994).
Nuclei of T24 bladder cells were stained either lightly or heavily
with hematoxylin and sperm cells were cytologically stained with
DIFF QUIK. Simultaneous staining with cytochemical stains such as,
for example, hematoxylin (blue/purple), methyl green, eosin (pink)
or DIF Quick (red) provides histological information and
contributes to multiparameter bright-field microscopic analysis.
Specimens were covered with mounting medium (obtained commercially)
under a coverslip.
Microscopy
A Leica DM microscope was equipped with a SD200 SpectraCube
(Applied Spectral Image, Migdal HaEmek, Israel) for acquisition of
spectral images. A halogen transmission light operating at 12 V for
daylight color temperature was used in the visible range (400-700
nm) by placing a WG360 UV cut-off filter and a BG38 infrared
cut-off filter in the illumination pathway. Neutral density filters
were used to optimize the light level for spectral imaging.
Spectral images were acquired with ASI acquisition software running
on a Dell Pentium PC. Typically, a spectral image is built of 200
frames of 300 ms with an interferometer stepsize angle of 15
degrees. Spectral analysis was performed on SpCube1.5 analysis
software (ASI).
Spectral imaging using the SD200 SpectraCube mounted on a
transmission light microscope allows for the measurement of the
absorption spectra of chromogenic dyes while retaining the spatial
information of the microscopic image. A spectral image is acquired
and for every pixel in the CCD image the absorption spectrum can be
retrieved. The so-called `optical density image` displays the
constituents of the specimen that absorb the light of certain
wavelengths. Regions that do not absorb light appear black. For
every pixel, an absorption curve can be produced, showing the
absorption intensities per wavelength. Pixel by pixel spectral data
can be utilized for subsequent mathematical operations. For
example, a spectrum-based classification would result instantly in
the pseudo-colorization of pixels with similar spectra. Defining
spectral signatures for specific regions within a specimen provides
flexibility for image analysis.
To demonstrate the improvement in clinical diagnosis provided by
the methods of the present invention as compared to techniques
available at the time the present invention was made, a comparison
was made between the bright field spectral imaging technology of
the present invention and state-of-the-art quantitative microscopy
software. For the latter procedure, a 3-chip color charged couple
device (CCD) video camera and Leica QWin software were used for
image capture and quantitative hue, as well as saturation and
intensity measurements. The hue value is a trivial but fixed number
for every color, whereas the saturation and intensity values vary
dependent on the quality of the detection. Color discrimination in
a 3-color video image therefore should be based on hue values. Hue
values are displayed in a histogram, showing the number of pixels
in an image for every hue value. Pixels with hue values which match
exactly can be selected and displayed with a single pseudo-color.
Hue-classification of all pixels in the image simultaneously is not
possible in a single operation.
For comparison of the spectral imaging method of the present
invention with quantitative microscopy, the same microscope was
equipped with a 3-chip color charged couple device camera (Sony,
Japan) controlled by QWin software (Leica Imaging, Cambridge UK),
for image acquisition and quantitative analysis, operating on a
Leica Q550 Pentium PC. Video images were acquired with a halogen
transmission light at 12 V (or 10.5V with a CB12 blue filter to
correct for daylight color temperature) and neutral density filters
for optimal video exposure times.
The in situ hybridization signals (spots) for centromere sequences
in T24 bladder cancer cells were analyzed after single-color
labeling, double-color labeling and triple-color labeling, with and
without cytological counterstaining. Single labeling experiments
without counterstain showed the spectra of the pure dyes. The
absorption spectra of the PO substrates DAB, TMB, and AEC and of
the AP substrates Fast Red, New Fuchsin and INT were measured,
showing specific spectral characteristics for each dye. Spectral
imaging of a triple-color in situ hybridization for chromosome
centromeres using TMB (green), New Fuchsin (red) and DAB (brown) as
reporter dyes resulted in good spectral separation of the
individual dyes (FIG. 1). Even the colors of small spots that could
not be easily discerned by eye were readily identified. The
absorption peaks were wide apart and the shapes of the curves
deviated clearly to allow for a spectrum-based color classification
of all spots.
In comparison, video images were acquired using a 3-chip color CCD
camera. Based on color hue values, the presence of the three colors
could be discriminated in a histogram. Hue measurement results,
however, could not be shown within the cellular context after a
single operation.
When cytological stains such as hematoxylin (blue purple) and DIF
Quick (red) are used, they are present throughout the cell or cell
compartment and are thus overlaying the hybridization spots. The
absorption spectrum of two co-localizing dyes seems to be additive,
meaning that the spectrum of the overlap is the sum of the two pure
spectra. The spectrum that is measured at the hybridization spots
is, therefore, mixed with the spectrum of the cytological stain.
The use of cytological stains, however, did not compromise the
separation of the absorption spectra of the reporter dyes (FIG. 2).
In single labeling experiments using New Fuchsin and heavy nuclear
staining with hematoxylin, the absorption spectrum of New Fuchsin
had shifted but this did not create a problem for the
classification of the hybridization signals.
In a double labeling experiment on sperm cells, using X and Y
chromosome-specific probes reported with, respectively, DAB and
TMB, and cytologically stained with DIF Quick, clear spectral
signatures of all three dyes can be defined (FIG. 3). Spectrum
based classification including all three dyes showed the
hybridization spots of X and Y in pseudo-colors which would have
been difficult from microscopic evaluation alone. The SpCube
analysis software provides for mathematical operations such as
spectrum subtraction and division. By selecting the average
spectrum of DIF Quick, a subtraction operation was executed,
eliminating the contribution of the cytological stain from the
spectral image. A similar effect can be achieved by division. The
classification image of FIG. 3 shows just the hybridization
spots.
In contrast, with the quantitative microscopy software, hue
measurements of single and double labeling experiments with
counterstaining were not consistently successful. Due to lower
color resolution, dyes of similar hue could not be discriminated in
the hue-histogram. Under influence of cytological stains, the hue
values of the hybridization spots shifted towards the hue of the
stain, which led to `drowning` of the spot in cases of intense
cytological staining or low hybridization signals. This phenomenon
could not be prevented because mathematical subtraction/division
operations can not be executed on these video images.
These data demonstrate that the bright field spectral imaging
method of the present invention provides for analysis of absorption
spectra with high precision while maintaining spatial information.
The use of cytological stains doesn't hamper spectral analysis and
thus greatly facilitates microscopic evaluation.
With quantitative microscopy, color discrimination based on hue
value using QWin software is possible by manually selecting spots.
However, cytological staining readily obscures color
discrimination. Using QWin software, the hue measurement results
can not be displayed together with spatial information in a single
operation. Thus, the data presented herein demonstrate that the
spectral imaging methods of the present invention provide higher
color resolution than 3-color video imaging, enabling the color
discrimination necessary for reliable and user-friendly
multi-parameter analysis of multi-color specimens.
Detection of Phenotypic and Genetic Markers According to the Method
of the Present Invention for Detection, Diagnosis, Characterization
and Prognosis of Cervical Cancer.
The diagnosis and staging of cancer is often not possible without
combining the results from several analyses. This includes: 1) the
interpretation of histomorphology after applying routine stains; 2)
the complementation of those histomorphological analyses with
pertinent genetic markers, such as the gain of 3q in cervical
cancers as definite identifiers of tumor progression; and 3) the
necessity to include phenotypic analysis by means of
immunohistochemistry with antibodies against commonly deregulated
oncogenes and tumor suppressor genes such as p53 and/or the
presence on viral genes such as human papilloma virus (HPV).
Multi-parameter analysis would benefit from the simultaneous
assessment of the above mentioned markers, which is possible, but
very difficult using fluorescence. Bright field with permanent dyes
comes with the described advantages, however, color discrimination
is a challenge. Spectral imaging overcomes these limitations by
allowing detection of multiple targets in pathological specimens
simultaneously.
As a specific example, in the progression of cervical carcinoma in
situ into invasive cervical carcinoma, an amplification of
chromosome region 3q24-28 is observed by comparative genomic
hybridization (CGH). This is the only detectable genetic event at
this stage of carcinogenesis and is therefore suitable as a
diagnostic marker for early-stage cervical carcinoma. CGH analyses
of late-stage cervical carcinomas revealed chromosomal gains in
regions of 2q and 5p. Cervical specimens can be biopsies, smears,
cytospin preparations, or sections of embedded cells. Comprehensive
diagnosis includes assessment of: 1) cytomorphology, using
morphological stains such as hematoxylin and eosin; 2) detection of
HPV genome by in situ hybridization; 3) screening for genotypic
markers 3q, 2p and 5p; 4) screening for tumor suppressor gene
products p53 and Rb, both known to interact with HPV antigens; and
5) screening for other phenotypic markers such as Ki67 and other
markers associated with the aggressiveness of tumors.
Although the present process has been described with reference to
specific details of certain embodiments thereof, it is not intended
that such details should be regarded as limitations upon the scope
of the invention except as and to the extent that they are included
in the accompanying claims.
Throughout this application, various publications are referenced.
The disclosures of these publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the state of the art to which this invention
pertains.
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